Checkpoint inhibitors block regulatory pathways that suppress T cell immunity either by modulating co- stimulation (anti-CTLA-4 Ab) or limiting responses of activated T cells in the tumor microenvironment (anti-PD- 1 Ab). The program cell death-1 (PD-1)/PD-L1 axis represents a mechanism of adaptive immune resistance that impedes anti-tumor immunity. Therapies that block the PD-1/PD-L1 pathway have shown ~30-40% response rates in patients with advanced cutaneous melanoma. Substantial evidence implicates tumor- reactive T cells as the targets of PD-1 blockade and, indeed, increased CD8+ T cell densities at the invasive tumor margin are strong correlates of clinical activity. T cells an recognize tumor-derived missense mutations translated as single amino acid substitutions within antigenic determinants (neoantigens) presented on HLA molecules by the cancer cell. Interestingly, response to checkpoint inhibitors correlates with higher mutational load in patients with melanoma and lung cancer implicating neoantigens as the targets of anti-tumor immunity. Knowledge of the neoantigen targets involved in anti-tumor immunity should foster a deeper understanding of how PD-1 blockade overcomes adaptive immune resistance. We previously reported that next generation sequencing, HLA binding prediction algorithms and laboratory assays allowed the identification of candidate melanoma neoantigens. Importantly incorporation of these candidates, as mutated peptides, in a dendritic cell (DC) vaccine increases the antigenic breadth and clonal diversity of neoantigen-specific CD8+ T cells in patients with metastatic melanoma. In the proposed proof-of-concept clinical trial, patients with advanced cutaneous melanoma will receive a personalized DC neoantigen vaccine sequentially followed by anti-PD-1 therapy. Characterization of neoantigen-specific T cell populations in blood and the tumor microenvironment (TME) will be performed to test the hypothesis that anti-PD-1 therapy inhibits adaptive immune resistance by enhancing neoantigen- specific CD8+ and CD4+ T cell immunity within the TME leading to more effective tumor regression. The hypothesis will be addressed in the experiments of the following Specific Aims: (1) To characterize neoantigen-specific T cell immunity elicited by a personalized DC vaccine formulation comprised of HLA class I and HLA-DR-restricted tumor-specific mutated peptides and (2) To characterize, in the peripheral blood and within the TME, the effect of anti-PD-1 antibody on vaccine-induced neoantigen-specific T cell immunity. This study will deliver new information regarding the feasibility and clinical benefit of targeting additional HLA class I alleles and HLA-DR restricted neoantigens by vaccination and provide a deeper understanding of the mechanisms by which PD-1 blockade enhances anti-tumor immunity in the TME. These findings should inform on the mechanisms of resistance for the ~60-70% of patients that currently fail anti-PD-1 monotherapy.